Protein tyrosine-tyrosine analogs and methods of using the same

ABSTRACT

PYY analogs are disclosed that include modifications that increase half-life when compared to native, human PYY, as well as additional modifications that increase potency and selectivity to the NPY2 receptor. Pharmaceutical compositions also are disclosed that include one or more of the PYY analogs described herein in a pharmaceutically acceptable carrier. Methods of making and using the PYY analogs also are disclosed, especially for treating obesity and obesity-related diseases and disorders such as type II diabetes mellitus.

The disclosure relates generally to biology and medicine, and moreparticularly it relates to Peptide Tyrosine-Tyrosine (PYY) analogs thatcan bind a neuropeptide Y (NPY) receptor such as the NPY2 receptor, aswell as compositions including the same and their therapeutic use intreating obesity and obesity-related diseases and disorders such as typeII diabetes (T2DM).

PYY is a member of the pancreatic polypeptide (PP) family and isinvolved in modulating food intake and energy expenditure following ameal (see, Tatemoto (1982) Proc. Natl. Acad. Sci. 79:2514-2518). PYY issecreted by L cells of the gastrointestinal track and has two mainendogenous forms—PYY₁₋₃₆ (SEQ ID NO: 1) and PYY₃₋₃₆ (SEQ ID NO:2).PYY₁₋₃₆ predominates over PYY₃₋₃₆ during fasting, whereas PYY₃₋₃₆predominates over PYY₁₋₃₆ following feeding. Dipeptidyl peptidase-IV(DPP-IV) hydrolyzes PYY₁₋₃₆ at a Pro²-Ile³ bond to produce PYY₃₋₃₆,which is more selective for the NPY2 receptor than PYY₁-36.

Plasma PYY₃₋₃₆ concentration typically increases within 15 minutes offood intake, peaks within 60-90 minutes, and remains elevated for up to6 hours before returning to baseline (see, Adrian et al. (1985)Gastroenterology 89:1070-1077; and De Silva & Bloom (2012) Gut Liver6:10-20). In this manner, PYY₃₋₃₆ is believed to impact appetite via itsdirect central effect and also via its effect on gut motility (i.e., itsanorectic effect). Additionally, PYY₃₋₃₆ is believed to mediate insulinsensitivity to thereby help lower blood glucose (i.e., its sensitizationeffect).

PYY₃₋₃₆ has been investigated as a potential therapeutic agent forbody-weight regulation in view of its anorectic effects, especially fortreating obesity and its associated diseases and disorders, includingT2DM and cardiovascular diseases (see, e.g., Intl. Patent ApplicationPublication No. 2002/47712; and Schwartz & Morton (2002) Nature418:595-597).

Unfortunately, exogenously administered PYY₃₋₃₆ has a short half-life(e.g., about 10-15 minutes) due to proteases and other clearancemechanisms (see, Lluis et al. (1989) Rev. Esp. Fisiol. 45:377-384; andTorang et al. (2016) Am. J. Physiol. Regul. Integr. Comp. Physiol.310:R866-R874), which presents challenges when using it as a therapeuticagent. With such a short half-life, PYY₃₋₃₆ should be administered atleast once-daily to exert a therapeutic effect, which is inconvenientfor an individual in need thereof. Efforts therefore have been made toincrease PYY₃₋₃₆'s half-life and/or to increase its NPY2 receptorselectivity. For example, Rubinstein et al. describe PYY analogs havinga 9-fluorenylmethoxy-carbonyl (Fmoc) or a2-sulfo-9-fluorenyl-methoxycarbonyl (FMS) radical to increase half-life(see, Intl. Patent Application Publication No. WO 2004/089279).Moreover, DeCarr et al. describe PYY analogs having amino-terminallylinked PEG moieties to increase half-life (see, DeCarr et al. (2007)Bioorg. Med. Chem. Lett. 17:1916-1919; see also, Ortiz et al. (2007) J.Pharmacol. Exp. Ther. 323:692-700). Furthermore, Kofoed et al. describePYY analogs having albumin-binding side chains, at least one modifiedresidue close to PYY's cleavage site (e.g. an N-methyl amino acid analogof an amino acid residue of interest), a N-glycine, and/or an argininemimetic to increase half-life (see, Intl. Patent Application PublicationNo. WO 2011/033068).

Despite significant increases in understanding PYY₃₋₃₆ role inmetabolism, there remains a need for additional PYY analogs, especiallyPYY analogs having improved potency and selectivity at the NPY2receptor.

As noted above, additional PYY analogs for therapeutic uses are needed.To address this need, the disclosure first describes PYY analogs thatinclude a base amino acid sequence of (with respect to the numbering ofnative, human PYY₁₋₃₆ (SEQ ID NO: 1)):³PKPEX₇PX₉X₁₀DASPEEX₁₇X₁₈RYYX₂₂X₂₃LRHYLNX₃₀LTRQRY³⁶ (Formula I), whereX₇ is any amino acid with a functional group available for conjugationand the functional group is conjugated to a C₁₆-C₂₂ fatty acid, X₉ is Eor G, X₁₀ is E or K, X₁₇ is L or W, X₁₈ is N or Q, X₂₂ is A or I, X₂₃ isE, D or S, and X₃₀ is E or W (SEQ ID NO:3), and where a carboxy-terminal(C-terminal) amino acid optionally is amidated.

In certain instances, the amino acid with the functional group availablefor conjugation at position X₇ can be C, D, E, K or Q. In particularinstances, the amino acid with the functional group available forconjugation at position X₇ is K, and the amino acid sequence can be oneof the following:

(SEQ ID NO: 4) ³PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY³⁶, (SEQ ID NO: 5)³PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY³⁶, (SEQ ID NO: 6)³PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY³⁶, (SEQ ID NO: 7)³PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY³⁶, or (SEQ ID NO: 8)³PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY³⁶.

In some instances, the C₁₆-C₂₂ fatty acid is conjugated to the aminoacid with the functional group available for conjugation via a linker.In certain instances, the C₁₆-C₂₂ fatty acid has a structure of—CO—(CH₂)_(a)—CO₂H, where a is an integer between 16 to 22. Inparticular instances, the fatty acid is a C₁₈ diacid or a C₂₀ diacidsuch as palmitic acid, stearic acid, arachidic acid or eicosanoic acid,especially a saturated C₁₈ diacid or C₂₀ diacid. Likewise, and in someinstances, the linker can be one or more units of[2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminohexanoic acid(Ahx), glutamic acid (E), γ-glutamic acid (γE) or combinations thereof.

In particular instances, the PYY analog can be one of the following:

In some instances, the base structure of the PYY analogs herein furthercan include the two amino-terminal (N-terminal) amino acids of native,human PYY₁₋₃₆ (SEQ ID NO:1), which subsequently can be processed in vivoto a PYY₃₋₃₆ analog (i.e., the N-terminal “YP” residues of SEQ ID NO: 1can be cleaved in vivo from any one of the PYY analogs).

In some instances, the PYY analogs have a charge of greater than −2,especially −3 or -4.

In some instances, the PYY analogs have a binding affinity at the humanNPY2 receptor that is greater than that of human PYY₃₋₃₆ (SEQ ID NO:2),such as from about 2-fold greater to about 10-fold greater, especiallyabout 2-fold greater to about 3-fold greater.

In some instances, the PYY analogs have a half-life that is longer thanthat of human PYY₃₋₃₆ (SEQ ID NO:2), such as from about 5 hours to about24 hours longer, especially about 12 hours.

Second, pharmaceutical compositions are described that include at leastone PYY analog herein or a pharmaceutically acceptable salt thereof(e.g., trifluroacetate salts, acetate salts or hydrochloride salts) anda pharmaceutically acceptable carrier. In some instances, thepharmaceutical compositions further can include carriers, diluentsand/or excipients.

Moreover, the pharmaceutical compositions can include an additionaltherapeutic agent such as, for example, other antidiabetic or weightloss agents, especially an incretin. In some instances, the incretin canbe glucagon (GCG) or a GCG analog. In other instances, the incretin canbe glucagon-like peptide-1 (GLP-1), GLP-1 (7-36)_(amide) or a GLP-1analog. In other instances, the incretin can be gastric inhibitorypeptide (GIP) or a GIP analog. In other instances, the incretin can be adual receptor agonist such as oxyntomodulin (OXM) or an OXM analog,GLP-1/GCG or GIP/GLP-1. In other instances, the incretin can be anincretin analog having triple receptor activity (i.e., incretin analogswith activity at each of the GIP, GLP-1 and GCG receptors). In otherinstances, the additional therapeutic agent can be a DPP-IV inhibitor.

Third, methods are described for using the PYY analogs herein,especially for using the PYY analogs to treat obesity andobesity-related diseases and disorders such as T2DM. The methods includeat least a step of administering to an individual in need thereof aneffective amount of a PYY analog as described herein or apharmaceutically acceptable salt thereof.

In some instances, the PYY analog can be subcutaneously (SQ)administered to the individual. Likewise, and in some instances, the PYYanalog can be administered daily, every other day, three times a week,two times a week, one time a week (i.e., weekly), biweekly (i.e., everyother week), or monthly. In certain instances, the PYY analog can beadministered SQ every other day, SQ three times a week, SQ two times aweek, SQ one time a week, SQ every other week, or SQ once a month. Inparticular instances, the PYY analog is administered SQ one time a week(QW).

Alternatively, the PYY analog can be orally administered to theindividual. As above, the PYY analog can be administered daily, everyother day, three times a week, two times a week, one time a week (i.e.,weekly), biweekly (i.e., every other week), or monthly. In certaininstances, the PYY analog can be administered orally every other day,orally three times a week, orally two times a week, orally one time aweek, orally every other week, or orally once a month. In particularinstances, the PYY analog is administered orally one time a week.

The methods also can include administering the at least one PYY analogin combination with an effective amount of an additional therapeuticagent such as a DPP-IV inhibitor or an incretin (e.g., GCG or a GCGanalog, GLP-1, GLP-1 (7-36)amide or a GLP-1 analog, GIP or a GIP analog,OXM or an OXM analog, GIP/GLP-1, GLP-1/GCG, or an incretin having triplereceptor activity). The DPP-IV inhibitor or incretin can be administeredsimultaneously, separately or sequentially with the PYY analog.

In some instances, the DPP-IV inhibitor or incretin can be administeredwith a frequency same as the PYY analog (i.e., every other day, twice aweek, or even weekly). In other instances, the DPP-IV inhibitor orincretin is administered with a frequency distinct from the PYY analog.In other instances, the DPP-IV inhibitor or incretin is administered QW.In still other instances, the PYY analog is administered SQ, and theDPP-IV inhibitor or the incretin can be administered orally.

In some instances, the individual is obese or overweight. In otherinstances, the individual is a person with diabetes (PwD), especiallyT2DM. In certain instances, the individual is obese with T2DM oroverweight with T2DM.

The methods also may include steps such as measuring or obtaining theindividual's weight and/or blood glucose and/or hemoglobin Alec (HbA1c)and comparing such obtained values to one or more baseline values orpreviously obtained values to assess the effectiveness of treatment.

The methods also may be combined with diet and exercise and/or may becombined with additional therapeutic agents other than those discussedabove.

Fourth, uses are described for the PYY analogs herein in treatingobesity and obesity-related diseases and disorders such as T2DM, whichoptionally can be administered simultaneously, separately orsequentially (i.e., in combination) with a DPP-IV inhibitor and/or anincretin such as GCG or a GCG analog, GLP-1, GLP-1 (7-36)_(amide) or aGLP-1 analog, GIP or a GIP analog, OXM or an OXM analog, GIP/GLP-1,GLP-1/GCG, or even an incretin having triple receptor activity.

Fifth, uses are described for the PYY analogs herein in manufacturing amedicament for treating obesity and obesity-related diseases anddisorders such as T2DM, where the medicament optionally may furtherinclude a DPP-IV inhibitor and/or an incretin such as GCG or a GCGanalog, GLP-1, GLP-1 (7-36)amide or a GLP-1 analog, GIP or a GIP analog,OXM or an OXM analog, GIP/GLP-1, GLP-1/GCG, or even an incretin havingtriple receptor activity.

One advantage of the PYY analogs herein is that they not only canfacilitate weight loss but also can lower glucose. In this manner,individuals, especially those susceptible to or having T2DM, can delayprogressing to exogenous insulin and can maintain target HbA1c goals.Moreover, the PYY analogs herein can enhance glycemic control byimproving insulin sensitization. Combined, GIP/GLP-1 and PYY analog canbe used for both glucose control (incretin+potential insulin sensitizer)and weight loss (synergistic). In particular, the PYY analogs herein cancause an up to about 12% weight loss alone when administered to anindividual in need thereof and can cause an up to about 25% weight lossin connection with an additional therapeutic agent such as an incretinwhen administered to an individual in need thereof.

Another advantage of the PYY analogs herein is that they can have ahalf-life of up to about 24 hours, thereby allowing for once weeklyadministration.

Another advantage of the PYY analogs herein is that they have increasedphysico-chemical stability and compatibility when compared to native,human PYY₃₋₃₆ (SEQ ID NO:2) and increased compatibility in a formulationwith incretins when compared to native, human PYY₃₋₃₆ (SEQ ID NO:2).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the disclosure pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the PYY analogs, pharmaceutical compositions andmethods, the preferred methods and materials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one element is present,unless the context clearly requires that there be one and only oneelement. The indefinite article “a” or “an” thus usually means “at leastone.”

Definitions

As used herein, “about” means within a statistically meaningful range ofa value or values such as, for example, a stated concentration, length,molecular weight, pH, sequence identity, time frame, temperature,volume, etc. Such a value or range can be within an order of magnitudetypically within 20%, more typically within 10%, and even more typicallywithin 5% of a given value or range. The allowable variation encompassedby “about” will depend upon the particular system under study, and canbe readily appreciated by one of skill in the art.

As used herein, “amino acid” means a molecule that, from a chemicalstandpoint, is characterized by the presence of one or more amine groupsand one or more carboxylic acid groups and may contain other functionalgroups. As is known in the art, there is a set of twenty amino acidsthat are designated as standard amino acids and that are used asbuilding blocks for most of the peptides/proteins produced by any livingbeing.

As used herein, “amino acid with a functional group available forconjugation” means any natural or unnatural amino acid with a functionalgroup that may be conjugated to a fatty acid by way of, for example, alinker. Examples of such functional groups include, but are not limitedto, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo andthiol groups. Additionally, examples of natural amino acids includingsuch functional groups include C (thiol), D (carboxyl), E (carboxyl), K(amino) and Q (amide).

As used herein, “analog” means a compound, such as a synthetic peptideor polypeptide, that activates a target receptor and that elicits atleast one in vivo or in vitro effect elicited by a native agonist forthat receptor.

As used herein, “anorectic effect” means an ability of the PYY analogsherein to reduce appetite, resulting in lower food consumption andultimately leading to weight loss. Anorectic effect also may refer to anability of the PYY analogs herein to increase gut motility.

As used herein, “C₁₆-C₂₂ fatty acid” means a carboxylic acid havingbetween 16 and 22 carbon atoms. The C₁₆-C₂₂ fatty acid suitable for useherein can be a saturated monoacid or a saturated diacid (“diacids” havea carboxyl group on each end).

As used herein, “AUC” means area under the curve.

As used herein, “effective amount” means an amount, concentration ordose of one or more PYY analogs herein, or a pharmaceutically acceptablesalt thereof which, upon single or multiple dose administration to anindividual in need thereof, provides a desired effect in such anindividual under diagnosis or treatment (i.e., may produce a clinicallymeasurable difference in a condition of the individual such as, forexample, a reduction in blood glucose, a reduction in HbA1c, and/or areduction in weight or body fat). An effective amount can be readilydetermined by one of skill in the art by using known techniques and byobserving results obtained under analogous circumstances. In determiningthe effective amount for an individual, a number of factors areconsidered, including, but not limited to, the species of mammal, itssize, age and general health, the specific disease or disorder involved,the degree of or involvement or the severity of the disease or disorder,the response of the individual, the particular PYY analog administered,the mode of administration, the bioavailability characteristics of thepreparation administered, the dose regimen selected, the use ofconcomitant medication, and other relevant circumstances.

As used herein, “half-maximal effective concentration” or “EC₅₀” means aconcentration of compound that results in 50% activation/stimulation ofan assay endpoint, such as a dose-response curve (e.g., cAMP).

As used herein, “in combination with” means administering at least oneof the PYY analogs herein either simultaneously, sequentially or in asingle combined formulation with one or more additional therapeuticagents.

As used herein, “incretin analog” means a peptide or polypeptide havingstructural similarities with, but multiple differences from, each ofGIP, GLP-1, GCG and OXM, especially native, human GIP, GLP-1, GCG andOXM. Some incretin analogs also have affinity for and activity at two oreven each of the GIP, GLP-1 and GCG receptors (i.e., agonist activity attwo receptors such as in OXM, GIP/GLP-1 or GLP-1/GCG, or even agonistactivity at all three receptors).

As used herein, “individual in need thereof” means a mammal, such as ahuman, with a condition, disease, disorder or symptom requiringtreatment or therapy, including for example, those listed herein. Inparticular, the preferred individual to be treated is a human.

As used herein, “long-acting” means that binding affinity and activityof a PYY analog herein continues for a period of time greater thannative, human PYY₁₋₃₆ (SEQ ID NO:1) and/or native, human PYY₃₋₃₆ (SEQ IDNO:2), allowing for dosing at least as infrequently as once daily oreven thrice-weekly, twice-weekly, once-weekly, or monthly. The timeaction profile of the PYY analogs herein may be measured using knownpharmacokinetic test methods such as those described in the Examplesbelow.

As used herein, “non-standard amino acid” means an amino acid that mayoccur naturally in cells but does not participate in peptide synthesis.Non-standard amino acids can be constituents of a peptide and oftentimes are generated by modification of standard amino acids in thepeptide (i.e., via post-translational modification). Non-standard aminoacids can include D-amino acids, which have an opposite absolutechirality of the standard amino acids above.

As used herein, “obese” or “obesity” means a condition in which anindividual has a body mass index (BMI) that is >30.0 kg/m². Seegenerally, “Overweight & Obesity” by the Center for Disease Control andPrevention available at cdc.gov/obesity/adult/defining.html; and“Definitions & Facts for Adult Overweight & Obesity” by the NationalInstitutes of Health atiddk.nih.gov/health-information/weight-management/adult-overweight-obesity/definition-facts.

As used herein, “obesity-related disease or disorder” means any diseasesor disorders that are induced/exacerbated by obesity including, but notlimited to, angina pectoris, cardiovascular disease, cholecystitis,cholelithiasis, congestive heart failure, dyslipidemia, fatty liverdisease, fertility complications, glucose intolerance, gout,hypertension, hypothyroidism, hyperinsulinemia, insulin resistance,osteoarthritis, polycystic ovary syndrome (PCOS), pregnancycomplications, psychological disorders, sleep apnea and otherrespiratory problems, stress urinary incontinence stroke, T2DM, uricacid nephrolithiasis (kidney stones), and cancer of the breast, colon,endometrium, esophagus, gall bladder, kidney, prostate and rectum.

As used herein, “overweight” means a condition in which an individualhas a BMI that is about 25.0 kg/m² to <30 kg/m². See, id.

As used herein, “PYY” means Peptide YY obtained or derived from anyspecies, such as a mammalian species, especially a human. PYY includesboth the native PYY (i.e., full-length) and variations thereof (i.e.,additions, deletions and/or substitutions of native PYY). Specific PYYsinclude, but are not limited to, native, human PYY₁₋₃₆ (SEQ ID NO:1) andnative, human PYY₃−0.3 (SEQ ID NO:2).

As used herein, “PYY analog” or “PYY analogs” means a PYY-like peptideor polypeptide that elicits one or more effects of native PYY at one ormore NPY receptors such as the NPY2 receptor. In some instances, the PYYanalogs herein can bind to a NPY receptor, especially the human NPY2receptor, with higher or lower affinity but demonstrate a longerhalf-life in vivo or in vitro when compared to native PYY, especiallyhuman PYY such as native, human PYY₁₋₃₆ (SEQ ID NO:1) and native, humanPYY₃₋₃₆ (SEQ ID NO:2). In this manner, the PYY analogs herein aresynthetic compounds that act as NPY2 receptor agonists.

As used herein, “saturated” means the fatty acid contains nocarbon-carbon double or triple bonds.

As used herein, “sensitizing effect” means an ability of the PYY analogsherein to increase the effect of insulin and thereby help lower bloodglucose.

As used herein, “treating” or “to treat” means attenuating, restraining,reversing, slowing or stopping progression or severity of an existingcondition, disease, disorder or symptom.

Certain abbreviations are defined as follows: “ACR” refers to urinealbumin/urine creatinine ratio; “amu” refers to atomic mass unit; “tBoc”refers to tert-butoxycarbonyl; “cAMP” refers to cyclic adenosinemonophosphate; “DMF” refers to dimethylformamide; “DMSO” refers todimethyl sulfoxide; “EIA/RIA” refers to enzymeimmunoassay/radioimmunoassay; “hr” refers to hour; “HTRF” refers tohomogenous time-resolved fluorescent; “IV” refers to intravenous; “kDa”refers to kilodaltons; “LC-MS” refers to liquid chromatography-massspectrometry; “MS” refers to mass spectrometry; “OtBu” refers toO-tert-butyl; “Pbf” refers toNG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; “RP-HPLC” refersto reversed-phase high performance liquid chromatography; “SQ” refers tosubcutaneous; “SEM” refers to standard error of the mean; “TFA” refersto trifluoroacetic acid; and “Trt” refers to Trityl.

PYY Analogs

The PYY analogs herein have structural similarities to, but manystructural differences, from native PYY peptides. For example, whencompared to native, human PYY₁₋₃₆ (SEQ ID NO:1) and/or native, humanPYY₃₋₃₆ (SEQ ID NO:2), the PYY analogs described herein includemodifications at one or more of positions 3, 7, 9, 10, 17, 18, 22, 23,30 and 31 with respect to the numbering of native, human PYY₁₋₃₆ (SEQ IDNO: 1). In certain instances, exemplary amino acid sequences of the PYYanalogs herein include (specific changes relative to correspondingresidue of native, human PYY (SEQ ID NO: 1) are in bold):

(SEQ ID NO: 3) ³ PKPEX ₇PX ₉ X ₁₀DASPEEX ₁₇ X ₁₈RYYX ₂₂ X ₂₃LRHYLNX ₃₀LTRQRY³⁶, (SEQ ID NO: 4) ³ PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY³⁶,(SEQ ID NO: 5) ³ PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY³⁶, (SEQ ID NO: 6) ³PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY³⁶, (SEQ ID NO: 7) ³PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY³⁶, and (SEQ ID NO: 8) ³PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY³⁶.

The PYY analogs herein result in sufficient activity at the human NPY2receptor but insufficient activity at the NPY1, NPY4 and NPY5 receptors.Likewise, the PYY analogs herein have beneficial attributes relevant totheir developability as therapeutic treatments, including improvedsolubility in aqueous solutions, improved chemical and physicalformulation stability, extended pharmacokinetic profile, and minimizedpotential for immunogenicity.

In some instances, the PYY analogs herein are amidated at a C-terminalamino acid to affect stability. In addition to the changes describedherein, the analogs may include one or more additional amino acidmodifications, provided, however, that the analogs remain capable ofbinding to and activating the human NPY2 receptor.

The PYY analogs herein further include a fatty acid conjugated, forexample, by way of a linker to a natural or unnatural amino acid with afunctional group available for conjugation (i.e., “acylation”). In someinstances, the amino acid with a functional group available forconjugation can be C, D, E, K and Q. In particular instances, the aminoacid with the functional group available for conjugation is K, whereconjugation is to an ε-amino group of a K side chain.

Here, acylation of the PYY analogs is at position 7 when compared tonative, human PYY₁₋₃₆ (SEQ ID NO:1). In this manner, the fatty acid canact as an albumin binder to provide for longer-acting analogs.

With respect to the fatty acid, it can be chemically conjugated to thefunctional group of the amino acid available for conjugation either by adirect bond or by a linker. The length and composition of the fatty acidimpacts the half-life of the PYY analogs, the in vivo potency of the PYYanalogs, and the solubility and stability of the PYY analogs.Conjugation to a C₁₆-C₂₂ saturated fatty monoacid or diacid therebyresults in PYY analogs that exhibit desirable half-life, desirable invivo potency, and desirable solubility and stability characteristics.

Exemplary saturated C₁₆-C₂₂ fatty acids for use herein include, but arenot limited to, hexadecanoic acid (i.e., palmitic acid, C₁₆ monoacid),hexadecanedioic acid (C₁₆ diacid), heptadecanoic acid (i.e., margaricacid, C₁₇ monoacid), heptadecanedioic acid (C₁₇ diacid), stearic acid(C₁₈ monoacid), octadecanedioic acid (C₁₈ diacid), nonadecylic acid(i.e., nonadecanoic acid, C₁₉ monoacid), nonadecanedioic acid (C₁₉diacid), eicosanoic acid (i.e., arachadic acid, C₂₀ monoacid),eicosanedioic acid (C₂₀ diacid), heneicosanoic acid (i.e., heneicosylicacid, C₂₁ monoacid), heneicosanedioic acid (C₂₁ diacid), docosanoic acid(i.e., behenic acid, C₂₂ monoacid), docosanedioic acid (C₂₂ diacid), andbranched and substituted derivatives thereof. In certain instances, theC₁₆-C₂₂ fatty acid can be a saturated Cis monoacid, a saturated C₁₈diacid, a saturated C₁₉ monoacid, a saturated C₁₉ diacid, a saturatedC₂₀ monoacid, a saturated C₂₀ diacid, and branched and substitutedderivatives thereof. In particular instances, the C₁₆-C₂₂ fatty acid canbe palmitic acid or hexadeconic acid, stearic acid or octadeconic acid,or arachidic acid or eicosanoic acid.

To assist in conjugating a fatty acid to a natural or unnatural aminoacid with the functional group available for conjugation, the PYYanalogs herein can include a linker. In some instances, the linker canbe at least one of AEEA, Ahx, E or γE, as well as combinations thereof.

When the linker includes the amino acids, it can have one to four E orγE amino acid residues. In some instances, the linker can include one ortwo E and/or γE amino acid residues. For example, the linker can includeeither one or two E and/or γE amino acid residues. In other instances,the linker can include one to four amino acid residues (such as, forexample, E or γE amino acids) used in combination with AEEA or Ahx.Specifically, the linker can be combinations of E and γE amino acidresidues with AEEA or Ahx. In still other instances, the linker can becombinations of one or two γE amino acid residues and one or two AEEA orAhx. In particular instances, the linker can be an (AEEA)₂·γE moiety, anAhx·E·γE moiety, or an AEEA·γE moiety.

Exemplary linker-fatty acid moieties can include (AEEA)₂·γE·C₂₀ diacid,Ahx·E·γE·C₁₈ diacid, or AEEA·γE·C₁₈ diacid. The structural features ofthese linker-fatty acid moieties result in analogs having improvedhalf-life when compared to native, human PYY₁₋₃₆ (SEQ ID NO: 1) ornative, human PYY₃₋₃₆ (SEQ ID NO:2).

Taken together, exemplary PYY analogs are:

Although the PYY analogs are described as having thirty-four amino acidslike that of native, human PYY₃₋₃₆ (SEQ ID NO:2), it is contemplatedthat the PYY analogs herein can have an amino acid sequence based uponnative, human PYY₁₋₃₆ (SEQ ID NO:1). That is, the PYY analogs caninclude the two N-terminal amino acids (i.e., the “YP” residues ofpositions 1 and 2 of SEQ ID NO: 1) of native, human PYY₁₋₃₆ (SEQ ID NO:1), which subsequently can be cleaved in vivo when administered to anindividual as would occur when native, human PYY₁₋₃₆ (SEQ ID NO: 1) isendogenously released.

Half-life of the PYY analogs herein may be measured using techniquesknown in the art including, for example, those described in the Examplesbelow. Likewise, affinity of the PYY analogs herein for each of thevarious human NPY receptors (e.g., NPY2R, NPY5R) may be measured usingtechniques known in the art for measuring receptor binding levelsincluding, for example, those described in the Examples below, and iscommonly expressed as an inhibitory constant (K_(i)) value. Moreover,activity of the PYY analogs herein at each of the receptors also may bemeasured using techniques known in the art, including, for example, thein vitro activity assays described below, and is commonly expressed asan EC₅₀ value.

As a result of the modifications described above, the PYY analogs hereinhave a half-life that is longer than that of native, human PYY₃₋₃₆ (SEQID NO:2). For example, the PYY analogs can have a half-life from about 5hours to about 24 hours, from about 6 hours to about 23 hours, fromabout 7 hours to about 22 hours, from about 8 hours to about 21 hours,from about 9 hours to about 20 hours, from about 10 hours to about 19hours, from about 11 hours to about 18 hours, from about 12 hours toabout 17 hours, from about 13 hours to about 16 hours, or even fromabout 14 hours to about 15 hours. Alternatively, the PPY analogs hereincan have a half-life that is about 5 hours, about 6 hours, about 7hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, or even about 24 hours,especially about 12 hours.

Likewise, the PYY analogs herein have a binding affinity at the NPY2receptor that is greater than that of native, human PYY₃₋₃₆ (SEQ IDNO:2), such as from about 2-fold to about 10-fold. Alternatively, thePYY analogs herein can have a binding affinity up to about 2-foldgreater, about 3-fold greater, about 4-fold greater, about 5-foldgreater, about 6-fold greater, about 7 fold-greater, about 8-foldgreater, about 9-fold greater, or even about 10-fold greater, especially2-fold greater to 3-fold greater, than that of native, human PYY₃₋₃₆(SEQ ID NO:2).

Pharmaceutical Compositions

The PYY analogs herein can be formulated as pharmaceutical compositions,which can be administered by parenteral routes (e.g., intravenous,intraperitoneal, intramuscular, subcutaneous or transdermal). Suchpharmaceutical compositions and techniques for preparing the same arewell known in the art. See, e.g., Remington, “The Science and Practiceof Pharmacy” (D. B. Troy ed., 21^(st) Edition, Lippincott, Williams &Wilkins, 2006). In particular instances, the PYY analogs areadministered SQ. Alternatively, however, the PYY analogs can beformulated in forms for other pharmaceutically acceptable routes suchas, for example, tablets or other solids for oral administration; timerelease capsules; and any other form currently used, including creams,lotions, inhalants and the like.

To improve their in vivo compatibility and effectiveness, the PYYanalogs herein may be reacted with any of a number of inorganic andorganic acids/bases to form pharmaceutically acceptable acid/baseaddition salts. Pharmaceutically acceptable salts and commonmethodologies for preparing them are well known in the art (see, e.g.,Stahl et al., “Handbook of Pharmaceutical Salts: Properties, Selectionand Use,” 2^(nd) Revised Edition (Wiley-VCH, 2011)). Pharmaceuticallyacceptable salts for use herein include sodium, trifluoroacetate,hydrochloride and acetate salts.

The PYY analogs herein may be administered by a physician orself-administered using an injection. It is understood the gauge sizeand amount of injection volume can be readily determined by one of skillin the art. However, the amount of injection volume can be ≤about 2 mLor even ≤about 1 mL, and the needle gauge can be ≥about 27 G or even≥about 29 G.

The disclosure also provides and therefore encompasses novelintermediates and methods useful for synthesizing the PYY analogsherein, or a pharmaceutically acceptable salt thereof. The intermediatesand PYY analogs herein can be prepared by a variety of methodologiesthat are well known in the art. For example, a method using chemicalsynthesis is illustrated in the Examples below. The specific syntheticsteps for each of the routes described may be combined in different waysto prepare the PYY analogs described herein. The reagents and startingmaterials are readily available to one of skill in the art.

The PYY analogs herein are generally effective over a wide dosage range.Exemplary doses of the PYY analogs herein or of pharmaceuticalcompositions including the same can be milligram (mg), microgram (μg),nanogram (ng) or picrogram (pg) amounts per kilogram (kg) of anindividual. In this manner, a daily dose can be from about 1 μg to about100 mg.

Here, the effective amount of the PYY analog in a pharmaceuticalcomposition can be a dose of about 0.25 mg to about 5.0 mg. One of skillin the art, however, understands that in some instances the effectiveamount (i.e., dose/dosage) may be below the lower limit of the aforesaidrange and be more than adequate, while in other cases the effectiveamount may be a larger doses and may be employed with acceptable sideeffects.

In addition to the PYY analog, the pharmaceutical composition also caninclude an additional therapeutic agent, especially other antidiabeticor weight loss agents. In some instances, the additional therapeuticagent can be at least one of an incretin or a DPP-IV inhibitor.Exemplary incretins include, but are not limited to, GCG, GLP-1, GLP-1(7-36)_(amide), GIP, OXM, a GCG analog, a GLP-1 analog, a GIP analog, anOXM analog, a GIP/GLP-1, a GLP-1/GCG, or even an incretin analog havingtriple receptor activity.

In this manner, the pharmaceutical composition can include an effectiveamount of a PYY analog of SEQ ID NO:9 and an incretin or a DPP-IVinhibitor, an effective amount of a PYY analog of SEQ ID NO:10 and anincretin or a DPP-IV inhibitor, an effective amount of a PYY analog ofSEQ ID NO: 11 and an incretin or a DPP-IV inhibitor, an effective amountof a PYY analog of SEQ ID NO:12 and an incretin or a DPP-IV inhibitor,or an effective amount of a PYY analog of SEQ ID NO:13 and an incretinor a DPP-IV inhibitor.

In those instances in which the incretin is GLP-1 or a GLP-1 analog, itcan be GLP-1 or a GLP-1 analog such as albiglutide, dulaglutide,liraglutide, semaglutide, or combinations thereof, especiallydulaglutide.

Methods of Making and Using the PYY Analogs

The PYY analogs herein can be synthesized via any number of peptidesynthesis methods known in the art using standard manual or automatedsolid-phase synthesis procedures. Automated peptide synthesizers arecommercially available from, for example, Applied Biosystems (FosterCity, Calif.) and Protein Technologies Inc. (Tucson, Ariz.). Reagentsfor solid-phase synthesis are readily available from commercial sources.Solid-phase synthesizers can be used according to the manufacturer'sinstructions for blocking interfering groups, protecting amino acidsduring reaction, coupling, deprotecting and capping of unreacted aminoacids.

Typically, an N-α-carbamoyl-protected amino acid and the N-terminalamino acid on the growing peptide chain attached to a resin are coupledat room temperature in an inert solvent such as DMF, N-methylpyrrolidoneor methylene chloride in the presence of coupling agents such asdiisopropyl-carbodiimide and 1-hydroxybenzotriazole. The N-α-carbamoylprotecting group is removed from the resulting peptide resin using areagent such TFA or piperidine, and the coupling reaction is repeatedwith the next desired N-α-protected amino acid to be added to thepeptide chain. Suitable amine protecting groups are well known in theart and are described, for example, in Green & Wuts, “Protecting Groupsin Organic Synthesis,” (John Wiley and Sons, 1991). The most commonlyused examples include tBoc and Fmoc. After completion of synthesis,peptides are cleaved from the solid-phase support with simultaneous sidechain deprotection using standard treatment methods under acidicconditions.

One of skill in the art will appreciate that the peptide chainsdescribed herein are synthesized with a C-terminal carboxamide. For thesynthesis of C-terminal amide peptides, resins incorporating Rink amideMBHA or Rink amide AM linkers typically are used with Fmoc synthesis,while MBHA resin is generally used with tBoc synthesis.

Crude peptides typically are purified using RP-HPLC on C8 or C18 columnsusing water-acetonitrile gradients in 0.05% to 0.1% TFA. Purity can beverified by analytical RP-HPLC. Identity of peptides can be verified byMS. Peptides can be solubilized in aqueous buffers over a wide pH range.

One use of the PYY analogs herein is for reducing blood glucose and/orbody weight in individuals, especially individuals who are overweight orobese and have T2DM. Administering a PYY analog as described herein canresult in glycemic control by improving insulin sensitization and weightloss. As such, the PYY analogs herein show glucose lowering efficacywith the added benefit of weight reduction, such that individuals candelay progression to insulin and maintain target HbA1c goals.

The methods can include the steps described herein, and these maybe be,but not necessarily, carried out in the sequence as described. Othersequences, however, also are conceivable. Moreover, individual ormultiple steps bay be carried out either in parallel and/or overlappingin time and/or individually or in multiply repeated steps. Furthermore,the methods may include additional, unspecified steps.

Such methods therefore can include selecting an individual who isoverweight and has T2DM or is predisposed to the same. Alternatively,the methods can include selecting an individual who is obese and hasT2DM or is predisposed to the same.

The methods also can include administering to the individual aneffective amount of at least one PYY analog as described herein, whichmay be in the form of a pharmaceutical composition as also describedherein. In some instances, the at least one PYY analog/pharmaceuticalcomposition can include an additional therapeutic agents such as anincretin or a DPP-IV inhibitor.

The concentration/dose/dosage of the at least one PYY analog andoptional incretin or DPP-IV inhibitor are discussed elsewhere herein.

With regard to a route of administration, the at least one PYY analog orpharmaceutical composition including the same can be administered inaccord with known methods such as, for example, orally; by injection(i.e., intra-arterially, intravenously, intraperitoneally,intracerebrally, intracerebroventricularly, intramuscularly,intraocularly, intraportally or intralesionally); by sustained releasesystems, or by implantation devices. In certain instances, the at leastone PYY analog or pharmaceutical composition including the same can beadministered SQ by bolus injection or continuously.

With regard to a dosing frequency, the at least one PYY analog orpharmaceutical composition including the same can be administered daily,every other day, three times a week, two times a week, one time a week(i.e., weekly), biweekly (i.e., every other week), or monthly. Incertain instances, the at least one PYY analog or pharmaceuticalcomposition including the same is administered SQ every other day, SQthree times a week, SQ two times a week, SQ one time a week, SQ everyother week or SQ monthly. In particular instances, the at least one PYYanalog or pharmaceutical composition including the same is administeredSQ one time a week (QW).

Alternatively, the at least one PYY analog or pharmaceutical compositionincluding the same can be orally administered. As above, and with regardto dosing frequency, the at least one PYY analog or pharmaceuticalcomposition including the same can be administered daily, every otherday, three times a week, two times a week, one time a week (i.e.,weekly), biweekly (i.e., every other week), or monthly. In certaininstances, the at least one PYY analog or pharmaceutical compositionincluding the same is administered orally every other day, orally threetimes a week, orally two times a week, orally one time a week, or orallyevery other week. In particular instances, the PYY analog isadministered orally one time a week.

With regard to those instances in which the at least one PYY analog orpharmaceutical composition including the same is administered incombination with an effective amount of an incretin, the incretin can beGCG or a GCG analog, GLP-1, GLP-1 (7-36)_(amide) or a GLP-1 analog, GIPor a GIP analog, OXM or an OXM analog, GIP/GLP-1, GLP-1/GCG, or even anincretin having triple receptor activity. The GCG, GCG analog, GLP-1,GLP-1 (7-36)_(amide), GLP-1 analog, GIP, GIP analog, OXM, OXM analog,GIP/GLP-1, GLP-1/GCG, or incretin having triple receptor activity can beadministered simultaneously, separately or sequentially with the atleast one PYY analog or pharmaceutical composition including the same.

Moreover, the GCG, GCG analog, GLP-1, GLP-1 (7-36)_(amide), GLP-1analog, GIP, GIP analog, OXM, OXM analog, GIP/GLP-1, GLP-1/GCG, orincretin having triple receptor activity can be administered with afrequency same as the at least one PYY analog or pharmaceuticalcomposition including the same (i.e., every other day, twice a week, oreven weekly). Alternatively, the GCG, GCG analog, GLP-1, GLP-1(7-36)_(amide), GLP-1 analog, GIP, GIP analog, OXM, OXM analog,GIP/GLP-1, GLP-1/GCG, or incretin having triple receptor activity can beadministered with a frequency distinct from the at least one PYY analogor pharmaceutical composition including the same. In other instances,the GCG, GCG analog, GLP-1, GLP-1 (7-36)_(amide), GLP-1 analog, GIP, GIPanalog, OXM, OXM analog, GIP/GLP-1, GLP-1/GCG, or incretin having triplereceptor activity is administered QW. In still other instances, the PYYanalog is administered SQ, and the GCG, GCG analog, GLP-1, GLP-1(7-36)_(amide), GLP-1 analog, GIP, GIP analog, OXM, OXM analog,GIP/GLP-1, GLP-1/GCG, or incretin having triple receptor activity can beadministered orally.

It is further contemplated that the methods may be combined with dietand exercise and/or may be combined with additional therapeutic agentsother than those discussed above.

EXAMPLES

The following non-limiting examples are offered for purposes ofillustration, not limitation.

Example 1: PYY Analog 1

One PYY analog incorporating the inventive concept can have a structureof:

Here, the N-terminus is free, and the C-terminal amino acid is amidatedas a C-terminal primary amide. The K at position 7 is chemicallymodified through conjugation to the ε-amino group of the K side chain atposition 7 with (Ahx-E-(γE)-CO—(CH₂)₁₆—COOH.

The PYY analog according to SEQ ID NO:9 is generated by solid-phasepeptide synthesis using Fmoc/t-Bu strategy on a SymphonyX AutomatedPeptide Synthesizer (PTI Protein Technologies Inc.) starting from RAPPAM-Rink Amide resin (H40023 Polystyrene AM RAM, Rapp polymere GmbH).Amino acid couplings are performed using 10 equivalents of amino acid,0.9 M diisopropylcarbodiimide (DIC) and 0.9 M Oxyma (1:1:1 molar ratio)in DMF for 3 h at 25° C. Deprotections are carried out using 25%piperidine solutions in DMF.

After elongating the peptide-resin as described above, the MTTprotecting group present in K at position 7 is removed using 30%Hexafluoroisopropanol (HFIP) in dichloromethane (DCM). Additionalcoupling/deprotection cycles using a Fmoc/t-Bu strategy to extend the Kat position 7 side chain involve Fmoc-6-aminohexanoic acid (Chem-ImpexInternational Catalog #02490), Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OH)-OtBu(ChemPep Catalog #100703) and HOOC—(CH₂)₁₆—COOtBu. In all couplings, 3equivalents of the building block are used with PyBOP (3 equiv) and DIEA(6 equiv) in DMF for 3 h at 25° C.

Concomitant cleavage from the resin and side chain protecting groupremoval are carried out in a solution containingTFA:triisopropylsilane:1,2-ethanedithiol:methanol:thioanisole 80:5:5:5:5(v/v) for 2 h at 25° C. followed by precipitation with cold ether. Crudepeptide is purified to >99% purity (15-20% purified yield) by RP-HPLC ona Phenyl Hexyl Column (Phenomenex, Luna; 5 μm, 100 A), where suitablefractions are pooled and lyophilized.

The purity of the PYY analog is examined by analytical RP-HPLC, andidentity is confirmed using LC/MS (observed: M+3H⁺/3=1659.2 (+/−0.2);calculated: M+3H⁺/3=1659.2; observed: M+4H⁺/4=1244.6 (+/−0.2);calculated: M+4H⁺/4=1244.6; observed: M+5H⁺/5=995.9 (+/−0.2);calculated: M+5H⁺/5=995.9).

Example 2: PYY Analog 2

One PPY analog incorporating the inventive concept can have a structureof:

As in Example 1, the N-terminus is free, and the C-terminal amino acidis amidated as a C-terminal primary amide. In contrast, however, the Kat position 7 is chemically modified through conjugation to the ε-aminogroup of the K side chain with([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γE)-CO—(CH₂)₁₈—COOH.

The PYY analog according to SEQ ID NO:10 is generated by solid-phasepeptide, similar to that described above in Example 1. Thus,FMOC-NHPEG2-CH₂COOH and HOOC—(CH₂)₁₈—COOtBu is attached to the sidechain after MTT cleavage using 3 equivalents of the building block withPyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

The purity of the PYY analog is examined by analytical RP-HPLC, andidentity is confirmed using LC/MS (observed: M+3H⁺/3=1665.4 (+/−0.2);calculated: M+3H⁺/3=1665.5; observed: M+4H⁺/4=1249.3 (+/−0.2);calculated M+4H⁺/4=1249.4; observed: M+5H⁺/5=999.7 (+/−0.2); calculated:M+5H⁺/5=999.7).

Example 3: PYY Analog 3

One PYY analog incorporating the inventive concept can have a structureof:

As in Example 1, the N-terminus is free, and the C-terminal amino acidis amidated as a C-terminal primary amide. In contrast, however, the Kat position 7 is chemically modified through conjugation to the ε-aminogroup of the K side chain with([2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γE)-CO—(CH₂)₁₆—COOH.

The PYY analog according to SEQ ID NO: 11 is generated by solid-phasepeptide, similar to that as described above in Example 1. Thus,FMOC-NHPEG2-CH₂COOH and HOOC—(CH₂)₁₆—COOtBu are attached to the sidechain after MTT cleavage using 3 equivalents of the building block withPyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

The purity of the PYY analog is examined by analytical RP-HPLC, andidentity is confirmed using LC/MS (observed: M+3H⁺/3=1664.7 (+/−0.2);calculated: M+3H⁺/3=1664.9; observed: M+4H⁺/4=1248.9 (+/−0.2);calculated: M+4H⁺/4=1248.9; observed: M+5H⁺/5=999.3 (+/−0.2);calculated: M+5H⁺/5=999.3).

Example 4: PYY Analog 4

One PYY analog incorporating the inventive concept can have a structureof:

As in Example 1, the N-terminus is free, and the C-terminal amino acidis amidated as a C-terminal primary amide. In contrast, however, the Kat position 7 is chemically modified through conjugation to the ε-aminogroup of the K side chain with([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γE)-CO—(CH₂)₁₆—COOH.

The PYY analog according to SEQ ID NO: 12 is generated by solid-phasepeptide, similar to that as described above in Example 1. Thus,FMOC-NHPEG2-CH₂COOH and HOOC—(CH₂)₁₆—COOtBu are attached to the sidechain after MTT cleavage using 3 equivalents of the building block withPyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

The purity of the PYY analog is examined by analytical RP-HPLC, andidentity is confirmed using LC/MS (observed: M+3H⁺/3=1664.8 (+/−0.2);calculated: M+3H⁺/3=1665.5; observed: M+4H⁺/4=1248.9 (+/−0.2);calculated: M+4H⁺/4=1249.4; observed: M+5H/5=998.9 (+/−0.2); calculated:M+5H/5=995.7).

Example 5: PYY Analog 5

One PYY analog incorporating the inventive concept can have a structureof:

As in Example 1, the N-terminus is free, and the C-terminal amino acidis amidated as a C-terminal primary amide. In contrast, however, the Kat position 7 is chemically modified through conjugation to the ε-aminogroup of the K side chain with([2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γE)₃—CO—(CH₂)₁₈—COOH.

The PYY analog according to SEQ ID NO: 13 is generated by solid-phasepeptide, similar to that as described above in Example 1. Thus,FMOC-NHPEG2-CH₂COOH and HOOC—(CH₂)₁₈—COOtBu are attached to the sidechain after MTT cleavage using 3 equivalents of the building block withPyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

The purity of the PYY analog is examined by analytical RP-HPLC, andidentity is confirmed using LC/MS (observed: M+3H⁺/3=1732.2 (+/−0.2);calculated: M+3H⁺/3=1732.3; observed: M+4H⁺/4=1299.4 (+/−0.2);calculated: M+4H⁺/4=1299.5; observed: M+5H⁺/5=1039.7 (+/−0.2);calculated: M+5H⁺/5=1039.8).

Example 6: In Vitro Activity of PYY Analogs

(1) In Vitro Binding to hNPY1, hNPY2, hNPY4, and hNPY5 Receptors

Purpose:

To assess the in vitro binding affinity (K_(i)) of the PYY analogs ofExamples 1 to 5 in the absence of bovine serum albumin (BSA) to thefollowing human (h) receptors: hNPY1R, hNPY2R, hNPY4R and hNPY5R.Competitive radioligand binding assays with membranes prepared from celllines overexpressing each of the recombinant receptors and the relevant[¹²⁵I]-labeled peptides are used in a scintillation proximity assay(SPA) method. The binding affinity for the associated native peptides,PYY₁₋₃₆ (SEQ ID NO: 1), PYY₃₋₃₆ (SEQ ID NO:2) and PancreaticPolypeptide₁₋₃₆ (PP₁₋₃₆; SEQ ID NO:14), is determined in each assay as acontrol.

Methods:

PYY analogs, native, human PYY₁₋₃₆ and control PYY₃₋₃₆ are synthesizedat Lilly Research Laboratories (Indianapolis, Ind., USA) and arecharacterized by LC/MS, NMR, and LC/UV analysis (99.5% purity). Peptidecontents are estimated at 80% powder mass. The peptides are prepared as10 mM stock solution in 100% DMSO and kept frozen at −20° C. until justprior to testing in the assays.

For hNPY1R, transient overexpression is performed using CHO cells.Stably transfected cell lines are prepared for hNPY2R and hNPY4R bysubcloning receptor cDNA into pcDNA3.1 expression plasmid andtransfecting into human embryonic kidney (HEK) 293 cells followed byselection with Geneticin. hNPY5R cloning is performed at Multispan, Inc.(Hayward, Calif.).

For the preparation of hNPY1R, hNPY2R, mNPY2R and hNPY4R crude cellmembranes, two different methods (described below) are utilized. hNPY5Rmembranes are purchased from Multipan, Inc. (# MCG1275).

Method 1—For hNPY2R and hNPY4R membranes, frozen cell pellets are lysedon ice in 10 mL hypotonic homogenization buffer containing 50 mM TrisHCl, pH 7.5, and Roche Complete™ Protease Inhibitors with EDTA(#1169749001) per gram of wet cell paste. The cell suspension isdisrupted using a glass Potter-Elvehjem homogenizer fitted with aTeflon® pestle for 25 strokes. The homogenate is centrifuged at 4° C. at1100× g for 10 minutes. The supernatant is collected and stored on icewhile the pellets are resuspended in homogenization buffer andrehomogenized as described above. The homogenate is centrifuged at1100×g for 10 minutes. The second supernatant is combined with the firstsupernatant and centrifuged at 35000×g for 1 hour at 4° C. The resultingmembrane pellet is resuspended in homogenization buffer containingprotease inhibitors at approximately 1 to 3 mg/mL, quick frozen inliquid nitrogen, and stored as aliquots in a −80° C. freezer until use.Protein concentration is determined using a BCA protein assay kit(Pierce, #23225) with BSA as a standard.

Method 2—For hNPY1R membranes, frozen cell pellets are lysed on ice in 5mL hypotonic homogenization Buffer containing 25 mM Tris HCl, pH 7.5, 1mM MgCl₂, 25 units/mL DNase I (Invitrogen, #18047-019) and RocheComplete™ Protease Inhibitors without EDTA (#11836170001) per gram ofwet cell paste. The cell suspension is disrupted using a glassPotter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes.The homogenate is centrifuged at 4° C. at 1800× g for 15 minutes in a 50mL conical tube. The supernatant is collected and stored on ice whilethe pellets are resuspended in homogenization buffer and rehomogenizedas described above, except DNase I is not used in the homogenizationbuffer. The homogenate is centrifuged at 1800× g for 15 minutes. Thesecond supernatant is combined with the first supernatant andcentrifuged at 25000×g for 30 minutes at 4° C. The resulting membranepellet is resuspended in homogenization buffer containing proteaseinhibitors at approximately 2 mg/mL, aliquoted, and stored in a −80° C.freezer until use. Protein concentration is determined using a BCAprotein assay kit (Pierce, #23225) with BSA as a standard.

General binding assay methods—The equilibrium dissociation constants(K_(d)) for the various receptor/radioligand interactions are determinedfrom saturation binding analysis using the same reagents and buffers asdescribed below for compound testing. The K_(d) values determined forthe receptor preparations used in this study are as follows: hNPY2R,0.0047 nM; hNPY1R, 0.07 nM; hNPY4R, 0.084 nM; and hNPY5R, 0.896 nM.

hNPY1R receptor binding protocol—The receptor binding affinity (K_(i))of PYY analog peptides and PYY₁₋₃₆ for hNPY1R is determined from acompetitive radioligand binding assay with human recombinant[¹²⁵I]-PYY₁₋₃₆ (# NEX341, 2200 Ci/mmol) obtained from Perkin Elmer(Waltham, Mass.). The assay is performed with a SPA method usingpolyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (#RPNQ0001, Perkin Elmer). Assay buffer (25 mM HEPES, pH 7.5, 1 mM MgCl₂,2.5 mM CaCl₂, and 0.2% w/v Bacitracin (RPI, #32000)) is used forpreparation of reagents. PYY analogs and PYY₁. 36 are thawed and 3-foldserially diluted in 100% DMSO (10 point concentration response curves)using a Tecan Evo liquid handler. A 20-fold step-down dilution ofpeptide into assay buffer is made to reduce the level of DMSO andpeptide concentration prior to addition into the assay plate. Next, 5 μLserially diluted peptide or DMSO is transferred into a Corning® 3632clear bottom assay plate containing 45 μL assay buffer or unlabeledPYY₁₋₃₆ control (nonspecific binding or NSB, at 10 nM finalconcentration). Then, 50 μL [¹²⁵I]-PYY₁₋₃₆ (0.05 nM final concentration)and 50 μL hNPY1R membranes (1.0 μg/well) are added. The final additionis 50 μL of WGA SPA beads (50 μg/well). Final DMSO concentration are0.125%. Plates are sealed and mixed on a plate shaker (setting 6) for 1minute and read with a PerkinElmer Trilux MicroBeta® scintillationcounter after 10 hours of incubation/bead settling time at roomtemperature. Final assay concentration ranges for peptides tested inresponse curves are: PYY analogs (2.5 μM to 0.13 nM) and PYY₁₋₃₆ (10 nMto 0.5 pM).

hNPY2 receptor binding protocol—The receptor binding affinity (K_(i)) ofPYY analog peptides and PP₁₋₃₆ for hNPY2R is determined from acompetitive radioligand binding assay as described above for hNPY1R.Final assay concentration ranges for peptides tested in response curvesare: PYY analogs (0.1 μM to 5 pM) and PYY₃₋₃₆ (10 nM to 0.5 pM).

hNPY4R receptor binding protocol—The receptor binding affinity (K_(i))of PYY analog peptides and PP₁₋₃₆ for hNPY4R is determined from acompetitive radioligand binding assay as described above for hNPY1R.Final assay concentration ranges for peptides tested in response curvesare: PYY analogs (2.5 μM to 0.13 nM) and PYY₁₋₃₆ (10 nM to 0.5 pM).

hNPY5R receptor binding protocol—The receptor binding affinity (K_(i))of PYY analog peptides and PYY₁₋₃₆ for hNPY5R is determined from acompetitive radioligand binding assay as described above for hNPY1R.Final assay concentration ranges for peptides tested in response curvesare: PYY analogs (1 μM to 10 pM) and PYY₁₋₃₆ (1 μM to 10 pM).

Data analysis for NPY receptor binding assays—Raw count per minute (CPM)data for concentration curves of PYY analogs, PYY₁₋₃₆, PYY₃₋₃₆, orPP₁₋₃₆ are converted to percent specific inhibition by subtractingnonspecific binding (NSB, binding in the presence of excess unlabeledPYY₁₋₃₆, PYY₃₋₃₆, or PP₁₋₃₆, respectively) from the individual CPMvalues and dividing by the total binding signal, also corrected bysubtracting nonspecific binding, as shown in the equation below:

${\% \mspace{14mu} {Specific}\mspace{14mu} {Inhibition}} = {100 - {\left\lbrack {\frac{{{CPM}\mspace{14mu} {for}\mspace{14mu} {Analog}\mspace{14mu} {or}\mspace{14mu} {Control}} - {{CPM}\mspace{14mu} {for}\mspace{14mu} {NSB}}}{{{CPM}\mspace{14mu} {for}\mspace{14mu} {Total}\mspace{14mu} {Binding}} - {{CPM}\mspace{14mu} {for}\mspace{14mu} {NSB}}} \times 100} \right\rbrack.}}$

Data are analyzed using four-parameter (curve maximum, curve minimum,IC₅₀, Hill slope) nonlinear regression routines (Genedata Screener,version 13.0.5, Genedata AG, Basal, Switzerland). The affinity (K_(i))is calculated from the relative IC₅₀ value based upon the equationK_(i)=IC₅₀/(1+D/K_(d)) where D=the concentration of radioligand in theexperiment, IC₅₀ is the concentration causing 50% inhibition of binding,and K_(d) is the equilibrium binding affinity constant of theradioligand determined from saturation binding analysis (listed above).A qualifier (>) indicates that the data did not reach 50% inhibition,compared to maximum binding in the absence of competitor, whereby theK_(i) is calculated using the highest concentration of the compoundtested in the assay.

Reported values for K_(i) are calculated as the geometric mean as shownbelow: Geometric Mean=10^((Arithmetic Mean of Log 10 Ki Values)).

Standard error of the mean (SEM) is calculated using the delta method asshown below:

${{SEM} = {{Geometric}\mspace{14mu} {Mean} \times \frac{{SD}\mspace{14mu} {of}\mspace{14mu} \log \mspace{11mu} {transformed}\mspace{14mu} {data}}{{Square}\mspace{14mu} {root}\mspace{14mu} {of}\mspace{14mu} n} \times {\ln (10)}}},$

where SD is the standard deviation, n is the number of independent runs,and ln(10) is the natural logarithm of 10.

Selectivity of peptides for hNPY2R (Y2) versus hNPY5R (Y5), hNPY4R (Y4),and/or hNPY1R (Y1) are calculated by dividing by the hNPY2R results innM.

Results:

TABLE 1 In Vitro Binding (K_(i)) to hNPY1R, hNPY2R, hNPY4R and hNPY5R.hNPY2R hNPY5R hNPY4R hNPY1R Fold Fold Fold Peptide (nM) (nM) (nM) (nM)(Y5/Y2) (Y4/Y2) (Y1/Y2) PP₁₋₃₆ — — 0.07 — — — — PYY₁₋₃₆ 0.007 0.37 50.06 52 714 8.6 PYY₃₋₃₆ 0.008 3.6 27 7 450 3375 875 Example 1 0.011 59626 256 5363 56909 23272 Example 20.070 >1000 >1840 >1460 >14285 >26286 >20857 Example 3 0.009 36.4112 >1550 4044 12444 >193750 Example 4 0.005 ND 280 >148 NA 56000 >29600Example 5 0.030 ND 560 >1530 NA 18666 >51000 Ref. 1* 0.016 189 — — 11812— — Ref. 2* 0.021 112 — — 5333 — — Ref. 3* 0.013 469 — — 36076 — —*Known PYY analog for comparison; see, Intl. Patent ApplicationPublication No. WO 2016/198682, where Ref. 1 corresponds to Compound 4therein, Ref. 2 corresponds to Compound 21 therein, and Ref. 3corres.00070011ponds to Compound 32 therein. ND - not detected NA - notapplicable

As shown above, the PYY analogs of Examples 1-5 are highly selective forthe hNPY2 receptor, even demonstrating reduced binding affinity tohNPY5, hNPY4 and hNPY1 receptors versus native, human PYY₃₋₃₆ (SEQ IDNO:2).

(2) In Vitro cAMP Activity on the Human NPY2 Receptor

Purpose:

To determine the in vitro functional activity of the PYY analogs ofExamples 1 to 5 compared to native, human PYY₃₋₃₆ by measuringinhibition of forskolin-induced intracellular cAMP production in HEK 293cells overexpressing the recombinant human NPY2 receptor.

Methods:

PYY analogs and human PYY₃₋₃₆ (SEQ ID NO:2) are synthesized,characterized and stored as described above in the receptor bindingassays.

Receptor cloning—A stably transfected cell line is prepared hNPY2receptor by subcloning receptor cDNA into pcDNA3.1 expression plasmidand transfecting it into HEK 293 cells followed by selection withGeneticin. Aliquots of cells (1×10⁷ cells/mL) at passage 9 are made andkept frozen in the vapor phase of a liquid nitrogen tank. These frozenaliquots are used at the time of the assay. Cells maintain greater than95% viability over several months.

hNPY2R cAMP assay—Inhibition of forskolin-induced cAMP production by PYYanalogs or PYY₃₋₃₆ is measured using HEK 293 cells overexpressingrecombinant hNPY2R. Frozen aliquots of cells are thawed in a 37° C.water bath. Cells are transferred to a 50 mL tube with 10 mL of culturemedium (MEM cell culture medium from Life Tech 11090-081 with 10% FBSfrom Life Tech 10082-147, 1 mM L-Glutamine from Life Tech 25030-081,1×NEAA from Life Tech 11140-050, 1 mM sodium pyruvate from Life Tech11360-070, 1× antibiotics-antimycotics from Life Tech 15240-062) and arecentrifuged 5 minutes at 1500 rpm in a Beckman tabletop centrifuge. Thesupernatant is removed and the cell pellet is resuspended in 10 mL ofcell culture medium followed by passage through a 40 μm strainer. Anaccurate count of cell number and cell viability is determined using aVi-Cell Analyzer from Beckman-Coulter (Vi-Cell XR 2.03). 8000 cells perwell are plated into a white 384 well assay plate (Corning,Poly-D-Lysine coated, white/opaque, cat #356661) using a Combi-TipDispenser (Thermo Scientific). Plates are centrifuged 1 second at 1000rpm and incubated 18 to 20 hours at 37° C. in a 5% CO₂-controlledincubator. Culture medium is removed from the assay plates by flickinggently on paper towels. 10 μL of assay buffer [1× HBSS (Hyclone, #SH3026801), 20 mM HEPES, pH 7.5 (Hyclone # SH30237.01), 0.1% w/v Casein(CTL Scientific Supply Corp., #440203H), 500 μM IBMX (Sigma-Aldrich#15876)] is added to the wells using the Combi-Tip Dispenser followed bycentrifugation for 10 seconds at 1500 rpm. A concentration responsecurve (20 point) at 2-fold dilutions is prepared in 100% DMSO usingacoustic dispensing technology (Labcyte Echo 550). The cells are treatedwith PYY analogs or human PYY₃. 36 for 45 minutes at 37° C. (final DMSOconcentration=1%), then stimulated with 1 μM forskolin (Sigma-Aldrich, #F6886) for 45 minutes at 37° C. The intracellular cAMP is quantifiedusing a CisBio cAMP-Gi Dynamic Kit (#62AM9PEB). Briefly, cAMP levelswithin the cell are detected using the HTRF kit reagents by addingcAMP-d2 conjugate in cell lysis buffer (10 μL) followed by adding theantibody anti-cAMP-Eu³⁺-Cryptate, also in cell lysis buffer (10 μL). Theresulting competitive assay is incubated for at least 60 minutes at roomtemperature, then read on a PerkinElmer Envision™ instrument withexcitation at 320 nm and emission at 665 nm and 620 nm. Final assayconcentration ranges for peptides tested in response curves are: PYYanalogs (0.1 μM to 0.2 pM) and human PYY₃₋₃₆ (10 nM to 0.02 pM). Astandard curve of known cAMP concentrations (0.5 μM to 1 pM) is preparedin assay buffer. Wells in the absence of added competitor or with anexcess of added human PYY₃₋₃₆ are included on each plate as MaximumResponse and Inhibitor Controls, respectively.

Data analysis for hNPY2 receptor cAMP assay—Time-resolved fluorescenceemission is used to calculate a fluorescence ratio (665 nM/620 nm),which is inversely proportional to the amount of cAMP present. Signalsfor PYY analogs and human PYY₃. 36 are converted to nM cAMP per wellusing a cAMP standard curve plotted as relative response units (emissionat 665 nm/620 nm*10,000, y-axis) versus concentration of cAMP (x-axis).

The amount of cAMP generated (nM) in each well is converted to a percentof the maximal response observed with forskolin only as shown in theequation below:

${\% \mspace{14mu} {Specific}\mspace{14mu} {Inhibition}} = \frac{{Peptide} - {{Inhibitor}\mspace{14mu} {Control}}}{{{Maximum}\mspace{14mu} {response}} - {{Inhibitor}\mspace{14mu} {control}}}$

where Inhibitor Control is the cAMP produced in the presence of addedexcess human PYY₃₋₃₆, Maximum Response is the cAMP produced in thepresence of forkolin only, and Peptide is the cAMP produced in thepresence of test peptide.

Percent specific inhibition (y-axis) is plotted against theconcentration of competitor (x-axis) and analyzed using a four-parameter(curve top, curve bottom, IC₅₀, Hill slope) nonlinear regression routine(Genedata Screener, version 13.0.5, Genedata AG, Basal, Switzerland) asdefined below:

$y = {{bottom} + {\frac{{top} - {bottom}}{1 + {\left( \frac{x}{{IC}\; 50} \right){Hill}\mspace{14mu} {Slope}}}.}}$

The relative IC₅₀ value represents the concentration causing 50%inhibition of forskolin-induced cAMP production.

Reported values for IC₅₀ are calculated as the geometric mean as shownbelow: Geometric Mean=10^((Arithmetic Mean of Log 10 IC50 Values)).

Standard error of the mean (SEM) is calculated using the delta method asshown below:

${{SEM} = {{Geometric}\mspace{14mu} {Mean} \times \frac{{SD}\mspace{14mu} {of}\mspace{14mu} \log \mspace{11mu} {transformed}\mspace{14mu} {data}}{{Square}\mspace{14mu} {root}\mspace{14mu} {of}\mspace{14mu} n} \times {\ln (10)}}},$

where SD is the standard deviation, n is the number of independent runs,and ln(10) is the natural logarithm of 10.

Results:

TABLE 2 In Vitro cAMP Activity on the hNPY2 Receptor. Peptide hNPY2REC₅₀ (nM) PYY₁₋₃₆ 0.12 PYY₃₋₃₆ 0.15 Example 1 0.07 Example 2 1.23Example 3 0.15 Example 4 0.06 Example 5 0.55 Ref. 1* 0.30 Ref. 2* 0.23Ref. 3* 0.32 *Known PYY analog for comparison; see, Intl. PatentApplication Publication No. WO 2016/198682, where Ref. 1 corresponds toCompound 4 therein, Ref. 2 corresponds to Compound 21 therein, and Ref.3 corresponds to Compound 32 therein.

As shown above, results from the cAMP assay demonstrate functionalactivity of the PYY analogs of Examples 1-5 on the hNPY2 receptor, withExample 2 showing the weakest potency at 12-fold lower potency versushuman PYY₃₋₃₆.

(3) In Vitro GTPγS Activity on the hNPR2 Receptor

Purpose:

To assess receptor-mediated activation of G-proteins by the PYY analogsof Examples 1 to 5. Receptor-mediated activation can be measured usingthe non-hydrolyzable GTP analog, GTPγ[³⁵S]. Agonist-mediated stimulationof G-protein-coupled receptors results in the activation ofmembrane-associated Gαβγ-protein heterotrimeric complexes. Thisrepresents a first step in transducing extracellular signals to modifyintracellular pathways. Herein, a GTPγ[³⁵S] functional assay is used toassess the potency of various PYY analogs at the hNPY2 receptor.

Methods:

PYY analogs, human PYY₁₋₃₆ (SEQ ID NO:1) and control peptide PYY₃₋₃₆(SEQ ID NO:2) are synthesized, characterized and stored as describedabove in the receptor binding assay.

For each test peptide, concentration response curves (CRC) with 1/3 logdilutions (logarithmic concentrations from −6.52 to −12.52) arecompleted with a Hamilton NIMBUS liquid handler in assay buffer (20 mMHEPES, pH 7.4, 100 mM NaCl, 6 mM MgCl₂, 1 mM EDTA) supplemented with0.2% Bacitracin (U.S. Biologicals #11805) and 0.5% DMSO. Final assayconcentrations of Bacitracin and DMSO are 0.05% and 0.125%,respectively. hNPY2R membranes (Multispan # HTS066M) are prepared to aconcentration of 7.5 ug/mL in assay buffer supplemented with 20 uM GDP(Sigma # G-7127) and 6 ug/mL Saponin (Sigma # S-4521) and hNPY2Rmembranes are incubated at room temperature for 20 minutes prior toaddition to the assay. GTPγS[³⁵S] (PerkinElmer # NEG030H) is prepared inassay buffer to a concentration of 0.6 nM. WGA SPA beads (PerkinElmer #RPNQ0001) are prepared at concentration of 12 mg/mL in assay buffer. Theassay is performed in a 96-well plate (Costar #3604) by first adding 100uL to the hNPY2R membranes, then 50 uL of CRC solution, then 50 uL ofGTPγS[³⁵S] solution for a final volume of 200 uL. The plate is coveredand placed on an orbital shaker (175 rpm for 45 minutes) at roomtemperature. Then, 25 uL of SPA beads are added and plate is re-sealedand vortexed to mix, then placed back on orbital shaker for 3 hours atroom temperature. The plate is then centrifuged for 5 minutes at 500 rpmand counted on a PerkinElmer 2450 Microplate counter for 1 minute perwell. Basal binding (CPM) is determined in the absence of PYY analog orhuman PYY₃₋₃₆ and is used to calculate a percent above basal value foreach concentration of peptide with the following equation: (PYY analog,or human PYY₃₋₃₆, CPM−Basal CPM)/(Basal CPM)*100. EC₅₀ (nM) values isdetermined by subjecting the logarithm of concentrations and percent ofbasal values to non-linear regression analysis (log(agonist) vs.response—Variable slope (four parameters)) in GraphPad Prism 7.0 usingthe equation: Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogEC₅₀−X)*HillSlope)). Geometric mean and standard error of the mean arecalculated from EC₅₀ (nM) values using the column statistics function inGraphPad Prism 7.0.

Results:

TABLE 3 In Vitro GTPγS Activity on the hNPY2 Receptor. Peptide hNPY2REC₅₀ (nM) PYY₁₋₃₆ 0.42 PYY₃₋₃₆ 0.43 Example 1 0.08 Example 2 1.83Example 3 0.06 Example 4 0.04 Example 5 0.16 Ref. 1* 0.22 Ref. 2* 0.13Ref. 3* 0.11 *Known PYY analog for comparison; see, Intl. PatentApplication Publication No. WO 2016/198682, where Ref. 1 corresponds toCompound 4 therein, Ref. 2 corresponds to Compound 21 therein, and Ref.3 corresponds to Compound 32 therein.

As shown above, results from the GTPγ[³⁵S] functional assay demonstrateactivity of the PYY analogs on the hNPY2 receptor, with Example 2showing the weakest potency at 4-fold lower potency versus humanPYY₃₋₃₆.

(4) Pharmacokinetics

Purpose:

To investigate the pharmacokinetic properties of the PYY analogs.

Methods:

LC/MS—Plasma concentrations of various PYY analogs are determined byLC/MS methods. The methods measure the whole compound; peptide pluslinked time extension. For the assay, PYY analogs and an internalstandard are extracted from mouse, rat or monkey plasma (50 μL) usingmethanol with 0.1% formic acid. The samples are centrifuged andsupernatant is transferred to a Thermo Protein Precipitation Plate. Thesamples are loaded on a Sep-Pak tC 18 SPE μElution Plate that isconditioned with methanol and 0.1% formic acid in water. The SPE columnsare washed twice with 0.1% formic acid in water. The compounds are theneluted using Formic Acid/Water/acetronitile (0.1:15:85), which are thendried and reconstituted prior to injecting an aliquot (10 μL) onto aThermo Acclaim PepMap100 C18, 300 μm×5 mm trap column and Thermo EasySpray PepMap C18, 75 μm×15 cm column for LC/MS analysis. The columneffluent is directed into a Thermo Q-Exactive Plus Mass Spectrometer fordetection and quantitation.

Pharmacokinetics of PYY analogs in CD-1 mice—The plasma pharmacokineticsof the PYY analogs are evaluated in male CD-1 mice following a singlesubcutaneous dose of 200 nmol/kg. Blood samples are collected from 2animals per time point over 168 hours. Since non-serial sampling is usedto evaluate the kinetics of the PYY analogs in mice, the meanconcentration versus time data are used to tabulate the pharmacokineticparameters for the PYY analogs following a single subcutaneous dose of200 nmol/kg. Plasma concentrations of PYY analogs are detected through120 hours following a single subcutaneous administration of 200 nmol/kg.

Pharmacokinetics of PYY analogs in SD rats—The plasma pharmacokineticsof the PYY analogs are evaluated in male Sprague Dawley rats following asingle subcutaneous dose of 50 nmol/kg. Blood samples are collected from2 animals per time point over 168 hours. Since serial sampling was usedto evaluate the kinetics of the PYY analogs in rats, individual animalconcentration versus time data were used to tabulate the pharmacokineticparameters for the PYY analogs following a single subcutaneous dose of50 nmol/kg. Plasma concentrations of PYY analogs are detected through120 hours following a single subcutaneous administration of 50 nmol/kg.

Pharmacokinetics of PYY analogs in cynomolgus monkeys—The plasmapharmacokinetics of the PYY analogs are evaluated in male and femalecynomolgus monkeys following a single subcutaneous dose of 50 nmol/kg.Blood samples are collected over 504 hours. Since serial sampling isused to evaluate the kinetics of the PYY analogs in monkeys, individualanimal concentration versus time data are used to tabulate thepharmacokinetic parameters for the PYY analogs following a singlesubcutaneous dose of 50 nmol/kg. Plasma concentrations of PYY analogsare detected through 504 hours following a single subcutaneousadministration of 50 nmol/kg.

Results:

TABLE 4 Mean Pharmacokinetic Parameters Following a Single SubcutaneousDose to Male CD-1 Mice. Dose T_(1/2) T_(max) C_(max) AUC_(inf) CL/FPeptide (nmol/kg) (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1200 15 6 1140 29795 6.71 Example 2 200 27 12 1014 56198 3.56 Example 3200 26 12 1360 52751 3.79 Example 4 200 15 12 916 31257 6.40Abbreviations: AUCinf = area under the curve from 0 to infinity; CL/F =clearance divided by bioavailability (F); Cmax = maximum concentration;Tmax = time at maximal concentration; T1/2 = half-life.

TABLE 5 Mean Pharmacokinetic Parameters Following a Single SubcutaneousDose to Male SD Rats. Dose T_(1/2) T_(max) C_(max) AUC_(inf) CL/FPeptide (nmol/kg) (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1 5022 12 221 8621 5.83 Example 3 50 38 12 399 21094 2.39 Example 4 50 19 8219 8289 6.41 Abbreviations: AUC_(inf) = area under the curve from 0 toinfinity; CL/F = clearance divided by bioavailability (F); C_(max) =maximum concentration; T_(max) = time at maximal concentration; T1/2 =half-life.

TABLE 6 Mean Pharmacokinetic Parameters Following a Single SubcutaneousDose to Cynomolgus Monkeys. Dose T_(1/2) T_(max) C_(max) AUC_(inf) CL/FPeptide (nmol/kg) (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1 50101 9 500 64552 0.775 Example 2 50 131 18 583 123173 0.408 Example 3 50148 9 599 105839 0.509 Abbreviations: AUC_(inf) = area under the curvefrom 0 to infinity; CL/F = clearance divided by bioavailability (F);C_(max) = maximum concentration; T_(max) = time at maximalconcentration; T1/2 = half-life.

Results:

These data demonstrate that the above compounds have a pharmacokineticprofile suitable for once weekly administration.

(5) Solubility & Stability

Purpose:

To determine the soluble pH ranges and stability of the PYY analogs

Methods:

Visual solubility range assessment—Lyophilized PYY analog powders arereconstituted in water at 4 mg/mL concentrations, and the pH is adjustedwith citric acid/phosphate buffer to pH 4. pH of the system is titratedup with 0.5 N NaCl to pH 8 and then titrated down with 0.5 N HCl to pH4.

Thermal stability evaluation—PYY analog solutions in 10 mM or 20 mMsodium phosphate, pH 7.0 at 1 mg/mL or 2 mg/mL concentration areprepared and incubated at 4′C and 40′C for 4 weeks. The samples at4-week time point are analyzed by size exclusion chromatography (SEC)and RP-HPLC.

SEC method—Performed using a TOSOH TSKgelG2000SW×1, 7.8 mm ID×30 cm, 5um column, with mobile phase composition of 50 mM sodium phosphate, 300mM NaCl, pH 7.0 with 20% acetonitrile over 30 minutes with a flow rateof 0.5 mL/min, λ—214 nm.

RP method—Performed using a Cortecs C18, 2.7 um, 4.6×50 mm column,20%-45% acetonitrile/water with 0.085% TFA over 10 minutes with a flowrate 1 mL/min, λ—214 nm.

Results:

TABLE 7 Solubility and Thermal Stability of the PYY Analogs. ThermalStability (10 or 20 mM Phosphate, pH 7.0) Solubility RP % Main Peak SEC% Main Peak Peptide (pH range) 4° C. 40° C. 4° C. 40° C. Example 1 >5.795.8 92.9 98.9 95.7 Example 2 >5.5 99.7 99.3 99.9 99.6 Example 3 >6.199.5 97.1 95.2 92.4 Example 4 >6.8 96.8 97.5 99.6 98.7 Example 5 >5.896.8 95.2 99.7 97.5

As shown above, all the peptides are soluble at pH >7.0. The stability,as assessed by RP and SEC, suggests that these peptides are relativestable under the aggressive thermal stress.

Example 7: In Vivo Effects of PYY

(1) In vivo effects on food intake and body weights in normal mice

Purpose:

To compare the effect of the PYY analogs of Examples 1 to 5 to reducebody weight and suppress food intake in normal mice after a singleinjection.

Methods:

Male C57Bl/6 mice from Envigo RMS (Indianapolis, Ind.) are maintained ona chow diet (5008; LabDiet, St. Louis, Mo.) and single housed in atemperature-controlled facility (74.0° F.; 23.3° C.) with a normal12:12-hour light cycle and free access to food and water. At 9-10 weeksof age, non-fasted body weights and initial food weights are recorded,and animals are administered a single subcutaneous injection of vehicleor peptide, followed by daily measurements of body weight and foodintake for 3 days post dose. Area under the curve analysis (AUC) iscalculated for both body weight and food intake versus vehicle. Example4 at 30 nmol/kg is used as benchmark of 100% efficacy for body weightand food intake in each run of the assay.

Results:

TABLE 8 Changes in Body Weight and Food Intake for 3 Days in C57/B16Mice Following a Single Dose of PYY Analog. Dose Δ Body Δ Food Peptide(nmol/kg, SC) Weight (%)** Intake (%)** Example 1 30 89 103 100 108 105Example 2 100 71 71 300 117 78 Example 3 30 98 93 100 111 100 Example 43 22 12 10 41 48 30 100 100 Example 5 100 70 99 300 129 118 Ref. 1* 10087 79 300 127 104 Ref. 2* 100 62 67 300 110 85 Ref. 3* 100 50 78 300 10499 *Known PYY analog for comparison; see, Inti. Patent ApplicationPublication No. WO 2016/198682, where Ref. 1 corresponds to Compound 4therein, Ref. 2 corresponds to Compound 21 therein, and Ref. 3corresponds to Compound 32 therein. **Example 4 at 30 nmol/kg (AUCs) setat 100% efficacy

As shown above, reductions in both body weight and food intakedemonstrate efficacy of the PYY analogs in vivo, where comparisons ondoses required for full efficacy demonstrate improvements in efficacy.

(2) In Vivo Effects on Food Intake and Body Weights in Diet-InducedObese Mice

Purpose:

To investigate the effect of daily dosing of the PYY analogs of Examples1-5 to reduce body weight, either alone or in combination with a GLP-1receptor agonist, over a two-week period in diet-induced obese (DIO)mice.

Methods:

DIO male C57Bl/6 mice (Taconic) at 20 weeks of age are maintained on a60% fat diet upon arrival (D12492; Research Diets, New Brunswick, N.J.).Animals are individually housed in a temperature-controlled facility(74.0° F.; 23.3° C.) with a 12-hour light/dark cycle (lights on 22:00)and free access to food and water. After a one-week acclimation periodof daily vehicle dosing, non-fasted body weights are measured, andanimals are randomized by body weight into experimental groups (n=6) andadministered daily subcutaneous injections of vehicle, a GLP-1 receptoragonist (GLP-1 RA; SEQ ID NO: 15), PYY analogs, or combinations of PYYanalogs plus the GLP-1 RA. After 2 weeks of dosing, non-fasted bodyweights are recorded and changes in average body weight versus vehicleare calculated. To determine additive or synergistic effects of PYYanalogs in combination with a GLP-1 RA, efficacy above that of the GLP-1RA alone (net effect) is calculated.

Results:

TABLE 9 Changes in Body Weight in a 2-Week Study in Diet- Induced ObeseMice with PYY Analogs Alone or in Combination with a GLP-1 ReceptorAgonist. Δ Body Weight (%) Dose PYY + Peptide (nmol/kg, SC) PYY AloneGLP-1RA* Example 1 1 −4 −18 3 −6 −26 10 −15 −31 Example 2 3 ND −1 10 ND−6 30 2 −11 Example 3 1 −3 −16 3 −6 −28 10 −23 −30 Example 4 0.3 0 −3 1−1 −12 3 −4 −17 10 −12 −21 Example 5 1 −2 −2 3 0 −11 10 −2 −18 30 −3 ND*Efficacy above that of a GLP-1 receptor agonist (GLP-1 RA)

As shown above, reductions in body weight with the PYY analogs, bothalone and in combination with the GLP-1 RA, demonstrate efficacy of thePYY analogs in vivo, where comparisons are made on the magnitude ofweight loss.

(3) In Vivo Effects on Body Weight and Glucose in Diabetic and Obese(db/db) Mice

Purpose:

To investigate the effect of daily dosing of the PYY analogs of Examples1 to 5 to reduce body weight and blood glucose levels over a ten-dayperiod in obese and diabetic mice (db/db).

Methods:

Lepr^(db/db) (db/db) male mice from Envigo RMS (Indianapolis, Ind.) aremaintained on a chow-style diet (5008; LabDiet, St. Louis, Mo.) andhoused 5 animals per cage in a temperature-controlled facility (74.0°F.; 23.3° C.) with a normal 12:12-hour light cycle and free access tofood and water. At 8-9 weeks of age, body weights and blood glucoselevels using Accu-Check® Glucometers (Roche Diabetes Care, Inc.,Indianapolis, Ind.) are measured, followed by daily subcutaneousinjections of vehicle or peptide. After 10 days of dosing, body weightsand blood glucose levels are measured and changes versus vehicletreatment are calculated.

Results:

TABLE 10 Effects on Body Weight and Blood Glucose in db/db Mice Treatedfor 10 Days. Dose Δ Body Weight Δ Glucose Peptide (nmol/kg, SC) (%) (%)Example 1 1 −1 7 3 −1 −10 10 −10 −56 30 −14 −59 Example 2 30 −5 −40 100−9 −60 300 −17 −67 Example 3 1 0 0 3 −5 −7 10 −9 −50 30 −15 −53 Example4 3 −2 −23 10 −12 −61 30 −17 −62 Example 5 10 −4 −24 30 −11 −59 100 −17−66

As shown above, reductions in body weight and blood glucose levels withthe PYY analogs demonstrate efficacy of the PYY analogs in vivo, wherecomparisons are made on the magnitude of weight loss and glucoselowering.

In conclusion, the PYY analogs herein show selectivity toward NPY2R.They also show dose-dependent reductions in body weight, as reflected innormal mice, diet-induced obese mice and db/db mice, as well asdose-dependent improvement in blood glucose in db/db mice, with the PYYanalogs of Examples 1, 3 and 4 being the most efficacious, in line withthe in vitro profile.

SEQUENCES SEQ ID NO: 1-Human PYY₁₋₃₆YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY SEQ ID NO: 2-Human PYY₃₋₃₆IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY SEQ ID NO: 3-PYY AnalogPKPEX₇PX₉X₁₀DASPEEX₁₇X₁₈RYYX₂₂X₂₃LRHYLNX₃₀LTRQRY SEQ ID NO: 4-PYY AnalogPKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY SEQ ID NO: 5-PYY AnalogPKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY SEQ ID NO: 6-PYY AnalogPKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY SEQ ID NO: 7-PYY AnalogPKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY SEQ ID NO: 8-PYY AnalogPKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY SEQ ID NO: 9-YY Analog

SEQ ID NO: 10-PYY Analog

SEQ ID NO: 11-PYY Analog

SEQ ID NO: 12-PYY Analog

SEQ ID NO: 13-PYY Analog

SEQ ID NO: 14-PP₁₋₃₆ APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY SEQ ID NO:15-GLP-1 RA HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LG

The invention claimed is:
 1. A Peptide Tyrosine-Tyrosine (PYY) analogcomprising an amino acid sequence of:PKPEX₇PX₉X₁₀DASPEEX₁₇X₁₈RYYX₂₂X₂₃LRHYLNX₃₀LTRQRY (Formula I), wherein X₇is any amino acid with a functional group available for conjugation andthe functional group is conjugated to a C₁₆-C₂₂ fatty acid, wherein X₉is E or G, wherein X₁₀ is E or K, wherein X₁₇ is L or W, wherein X₁₈ isN or Q, wherein X₂₂ is A or I, wherein X₂₃ is E, D or S, wherein X₃₀ isE or W (SEQ ID NO:3), and wherein a C-terminal amino acid is optionallyamidated.
 2. The PYY analog of claim 1, wherein X₇ is selected from thegroup consisting of C, D, E, K and Q.
 3. The PYY analog of claim 1,wherein X₇ is K and conjugation to the C₁₆-C₂₂ fatty acid is through anepsilon-amino group of a K side chain.
 4. The PYY analog of claim 1,wherein the amino acid sequence is selected from the group consistingof: (SEQ ID NO: 4) PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY; (SEQ ID NO: 5)PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY; (SEQ ID NO: 6)PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY; (SEQ ID NO: 7)PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY; and (SEQ ID NO: 8)PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY.


5. The PYY analog of claim 1, wherein the C₁₆-C₂₂ fatty acid is selectedfrom the group consisting of a hexadecanoic acid, a hexadecanedioicacid, a heptadecanoic acid, a heptadecanedioic acid, a stearic acid, anoctadecanedioic acid, a nonadecylic acid, a nonadecanedioic acid, aneicosanoic acid, an eicosanedioic acid, a heneicosanoic acid, aheneicosanedioic acid, a docosanoic acid, a docosanedioic acid, andbranched and substituted derivatives thereof.
 6. The PYY analog of claim5, wherein the C₁₆-C₂₂ fatty acid is a C₁₈-C₂₀ fatty acid.
 7. The PYYanalog of claim 6, wherein the C₁₈-C₂₀ fatty acid is a straight-chainfatty acid having a formula of CO—(CH₂)_(x)-CO₂H, and wherein _(x) is 18or
 20. 8. The PYY analog of claim 7, wherein the C₁₈-C₂₀ fatty acid isselected from the group consisting of palmitic acid, stearic acid,arachidic acid and eicosanoic acid.
 9. The PYY analog of claim 1,wherein the C₁₆-C₂₂ fatty acid is conjugated to the amino acid with thefunctional group available for conjugation via a linker.
 10. The PYYanalog of claim 9, wherein the linker can be one or more units selectedfrom the group consisting of [2-(2-amino-ethoxy)-ethoxy)]-acetic acid(AEEA), aminohexanoic acid (Ahx), glutamic acid (E), gamma glutamic acid(γE) and combinations thereof.
 11. The PYY analog of claim 1, whereinthe amino acid sequence is:


12. The PYY analog of claim 1, wherein the amino acid sequence is:


13. The PYY analog of claim 1, wherein the amino acid sequence is:


14. The PYY analog of claim 1, wherein the amino acid sequence is:


15. The PYY analog of claim 1, wherein the amino acid sequence is:


16. A pharmaceutical composition comprising: at least one PeptideTyrosine-Tyrosine (PYY) analog of claim 1 or a salt thereof; and one ormore pharmaceutically acceptable carriers, diluents and excipients. 17.The pharmaceutical composition of claim 16 further comprising anadditional therapeutic agent.
 18. The pharmaceutical composition ofclaim 17, wherein the additional therapeutic agent is an incretinselected from the group consisting of glucagon (GCG), a GCG analog,glucagon-like peptide-1 (GLP-1), GLP-1_(7-36-amide), a GLP-1 analog,gastric inhibitory peptide (GIP), a GIP analog, oxyntomodulin (OXM), anOXM analog, a GIP/GLP-1, a GLP-1/GCG, or an incretin analog havingtriple receptor activity.
 19. The pharmaceutical composition of claim17, wherein the additional therapeutic agent is a dipeptidylpeptidase-IV (DPP-IV) inhibitor.
 20. A method of treating obesity or anobesity related disease or disorder, the method comprising a step of:administering to an individual in need thereof an effective amount of aPeptide Tyrosine-Tyrosine (PYY) analog of claim 1 or a pharmaceuticallyacceptable salt thereof.
 21. The method of claim 20, wherein the PYYanalog or pharmaceutically acceptable salt thereof is subcutaneously(SQ) administered to the individual.
 22. The method of claim 20, wherethe PYY analog or pharmaceutically acceptable salt thereof is orallyadministered to the individual.
 23. The method of claim 20, wherein thePYY analog or pharmaceutically acceptable salt thereof is administereddaily, every other day, three times a week, two times a week, one time aweek, or biweekly.
 24. The method of claim 20, wherein the PYY analog orpharmaceutically acceptable salt thereof is administered SQ one time aweek (QW).
 25. The method of claim 20, wherein the PYY analog orpharmaceutically acceptable salt thereof is administered orally one timea week.
 26. The method of claim 20 further comprising administering anadditional therapeutic agent.
 27. The method of claim 26, wherein theadditional therapeutic agent is an incretin selected from the groupconsisting of glucagon (GCG), a GCG analog, glucagon-like peptide-1(GLP-1), GLP-1_(7-36-amide), a GLP-1 analog, gastric inhibitory peptide(GIP), a GIP analog, oxyntomodulin (OXM), an OXM analog, a GIP/GLP-1, aGLP-1/GCG, or an incretin analog having triple receptor activity. 28.The method of claim 26, wherein the additional therapeutic agent is adipeptidyl peptidase-IV (DPP-IV) inhibitor.